Capture, identification and use of a new biomarker of solid tumors in body fluids

ABSTRACT

A new sensitive cell biomarker of solid tumors is identified in blood. This biomarker can be used to determine presence of carcinomas, rapid determination of treatment response, early detection of cancer, early detection of cancer recurrence, and may be used to determine therapy.

BACKGROUND

Field of the Invention

The present invention relates generally to identification of a biomarkerin blood and other body fluids that can be used for detection of solidtumors and to monitor efficacy of chemotherapy and radiation therapytreatments. The present invention also relates to the use of thebiomarker in combination with circulating tumor cells, free plasma andserum DNA cancer markers, cancer-associated protein markers and otherbiomarkers.

Related Art

When tumor cells break away from primary solid tumors, they penetrateinto the blood or lymphatic circulation, and ultimately leave the bloodstream and enter other organ or tissue to form metastasis. 90% ofcancer-related deaths are caused by the metastatic process. The mostcommon metastatic sites are the lung, liver, bone and brain. Tumor cellsfound in the circulation are called circulating tumor cells (CTCs). Manyresearch publications and clinical trials show that CTCs have clinicalutility (i) by providing prognostic survival and cancer recurrenceinformation by counting the number of cells in the blood stream, and(ii) by providing treatment information by looking at proteinexpressions, gene mutations and translocations in the CTCs. However,CTCs cannot be found consistently, even in stage IV patients.

Some medical conditions may be diagnosed by detecting the presence ofcertain types of cells in bodily fluid. In particular, cells indicativeor characteristic of certain medical conditions may be larger and/orless flexible than other cells found in certain bodily fluids.Accordingly, by collecting such larger and/or less flexible cells from aliquid sample of a bodily fluid, it may be possible to diagnose amedical condition based on the cells collected.

SUMMARY

The present invention is directed to and discloses a type of cell withspecial characteristics that is found in the blood of cancer patients.The cell type, termed “circulating Cancer Associated Macrophage-Likecell” (CAML), is described herein and shown to be associated with thepresence of solid tumors in a patient. This cell type is shown by datapresented herein to have clinical utility in that it can be used as abiomarker for a variety of medical applications. This cell type has beenfound consistently in the peripheral blood of stage I to stage IV cancerof epithelial origin by microfiltration using precision microfilters.

CAMLs can be used as a biomarker to provide a diagnosis of cancer, inparticular, an early detection of cancer, an early detection of cancerrecurrence, and a determination of cancer mutation. CAMLs can also beused as a biomarker in determining appropriate courses of therapy, inparticular, the cells can be used in a rapid determination ofeffectiveness of chemotherapy and radiation therapy treatment response.

CAMLS may be used independently as a cancer marker, and CAMLS can beused together with CTCs, free DNAs, proteins and other biomarkers inbody fluids to provide a more complete understanding of the patient'sdisease.

More specifically, and in a first embodiment, the present invention isdirected to methods of screening a subject for cancer, comprisingdetecting circulating cancer associated macrophage-like cells (CAMLs) ina biological sample from a subject. In particular aspects, when CAMLsare detected in the biological sample, the subject is identified aspotentially having a carcinoma or solid tumor. In other aspects, whenCAMLs are detected in the biological sample, the subject is identifiedas having a carcinoma or solid tumor. In certain aspects, the methodsencompassed by this embodiment also include detecting circulating tumorcells (CTCs) in the biological sample. In particular aspects of thisfirst embodiment, the subject is a subject suspected of having cancer.

In a second embodiment, the present invention is directed to methods fordiagnosing cancer in a subject, comprising detecting CAMLs in abiological sample from a subject, wherein when CAMLs are detected in thebiological sample, the subject is diagnosed with cancer. In certainaspects, the methods encompassed by this embodiment also includedetecting CTCs in the biological sample, wherein when CAMLs and CTCs aredetected in the biological sample, the subject is diagnosed with cancer.

In a third embodiment, the present invention is directed to methods fordetecting recurrence of cancer in a subject, comprising detecting CAMLsin a biological sample from a subject previously treated for cancer,wherein when CAMLs are detected in the biological sample, recurrence ofcancer is detected. In certain aspects, the methods encompassed by thisembodiment also include detecting CTCs in the biological sample, whereinwhen CAMLs and CTCs are detected in the biological sample, recurrence ofcancer is detected.

In a fourth embodiment, the present invention is directed to methods forconfirming a diagnosis of cancer in a subject, comprising detectingCAMLs in a biological sample from a subject diagnosed with cancer,wherein when CAMLs are detected in the biological sample, a diagnosis ofcancer is confirmed in the subject. In certain aspects, the methodsencompassed by this embodiment also include detecting CTCs in thebiological sample, wherein when CAMLs and CTCs are detected in thebiological sample, a diagnosis of cancer is confirmed in the subject. Inparticular aspects, the initial cancer diagnosis is via mammography, PSAtest, or presence of CAl25. In a particular aspect, the subject issuspected of having cancer.

In aspects of the first through fourth embodiments, CAMLs are detectedusing one or more means selected from the group consisting of sizeexclusion methodology, red blood cell lysis, FICOLL, a microfluidicchip, and flow cytometry, or a combination thereof. In particularaspects, the size exclusion methodology comprises use of a microfilter.Suitable microfilters can have a variety of pore sizes and shapes.Microfilters having pores of about 7-8 microns in size are acceptable,and include round and rectangular pore shapes. Microfilters having roundpores of about 7-8 microns in size are especially optimal when polymericmicrofilters are used. In a preferred aspect, the microfilter hasprecision pore geometry and uniform pore distribution.

In certain aspects of the first through fourth embodiments, CAMLs andCTCs are simultaneously detected using a microfilter. Suitablemicrofilters can have a variety of pore sizes and shapes. Microfiltershaving pores of about 7-8 microns in size are acceptable, and includeround and rectangular pore shapes. Microfilters having round pores ofabout 7-8 microns in size are especially optimal when polymericmicrofilters are used. In a preferred aspect, the microfilter hasprecision pore geometry and uniform pore distribution.

In certain aspects of the first through fourth embodiments, CAMLs aredetected using a microfluidic chip based on physical size-based sorting,hydrodynamic size-based sorting, grouping, trapping, immunocapture,concentrating large cells, or eliminating small cells based on size.

In certain aspects of the first through fourth embodiments, CAMLs aredetected using a CellSieve™ low-pressure microfiltration assay.

In aspects of the first through fourth embodiments, the biologicalsample is one or more selected from the group consisting of peripheralblood, blood, lymph nodes, bone marrow, cerebral spinal fluid, tissue,and urine. In a preferred aspect, the biological sample is peripheralblood. In other aspects, the blood is antecubital-vein blood,inferior-vena-cava blood or jugular-vein blood.

In aspects of the first through fourth embodiments, the cancer is one ormore of a solid tumor, Stage I cancer, Stage II cancer, Stage IIIcancer, Stage IV cancer, epithelial cell cancer, breast cancer, prostatecancer, lung cancer, pancreatic cancer, and colorectal cancer.

In a fifth embodiment, the present invention is directed to methods formonitoring efficacy of a cancer treatment, comprising (a) determiningthe number of CAMLs in a biological sample from a subject before cancertreatment, and (b) comparing the number of CAMLs determined in (a) to anumber of CAMLs determined from a similar biological sample from thesame subject at one or more time points after treatment. In certainaspects, the methods further comprise (c) determining the number of CTCsin the biological sample of (a), and (d) comparing the number of CTCsdetermined in (c) to a number of CTCs determined from the biologicalsample of (b).

In aspects of the fifth embodiment, a change in the number of CAMLs isan indication of treatment efficacy, where the change is an increase ora decrease in the number of CAMLs.

In aspects of the fifth embodiment, the cancer treatment is chemotherapyor radiation therapy.

In aspects of the fifth embodiment, the number of CAMLs is determinedusing one or more means selected from the group consisting of sizeexclusion methodology, red blood cell lysis, FICOLL, a microfluidicchip, and flow cytometry, or a combination thereof. In a particularaspect, the size exclusion methodology comprises use of a microfilter.Suitable microfilters can have a variety of pore sizes and shapes.Microfilters having pores of about 7-8 microns in size are acceptable,and include round and rectangular pore shapes. Microfilters having roundpores of about 7-8 microns in size are especially optimal when polymericmicrofilters are used. In a preferred aspect, the microfilter hasprecision pore geometry and uniform pore distribution. In a particularaspect, the number of CAMLs is determined using a microfluidic chipbased on physical size-based sorting, hydrodynamic size-based sorting,grouping, trapping, immunocapture, concentrating large cells, oreliminating small cells based on size. In a particular aspect, thenumber of CAMLs is determined using a CellSieve™ low-pressuremicrofiltration assay.

In certain aspects of the fifth embodiment, the numbers of CAMLs andCTCs are determined simultaneously using a microfilter. Suitablemicrofilters can have a variety of pore sizes and shapes. Microfiltershaving pores of about 7-8 microns in size are acceptable, and includeround and rectangular pore shapes. Microfilters having round pores ofabout 7-8 microns in size are especially optimal when polymericmicrofilters are used. In a preferred aspect, the microfilter hasprecision pore geometry and uniform pore distribution.

In aspects of the fifth embodiment, the biological sample is one or moreselected from the group consisting of peripheral blood, blood, lymphnodes, bone marrow, cerebral spinal fluid, tissue, and urine. In apreferred aspect, the biological sample is peripheral blood. In otheraspects, the blood is antecubital-vein blood, inferior-vena-cava bloodor jugular-vein blood

In aspects of the fifth embodiment, the cancer is one or more of a solidtumor, Stage I cancer, Stage II cancer, Stage III cancer, Stage IVcancer, epithelial cell cancer, breast cancer, prostate cancer, lungcancer, pancreatic cancer, and colorectal cancer.

In a sixth embodiment, the present invention is directed to an isolatedcirculating cancer associated macrophage-like cell (CAML), wherein thecell has the following characteristics: (a) large atypical nucleushaving a size of about 14-64 μm; (b) expression of one or more ofcytokeratin 8, 18 and 19, wherein the cytokeratin is diffused, orassociated with vacuoles and/or ingested material; (c) cell size rangingfrom about 20 micron to about 300 microns; (d) morphological shapeselected from the group consisting of spindle, tadpole, round, oblong,and amorphous; and (e) CD45 positive phenotype. In one aspect of thisembodiment, the cell has one or more of the following additionalcharacteristics: (f) expression of diffuse EpCAM with nearly uniformdistribution; (g) expression of one or more markers of a primary tumor;(h) expression of monocytic CD11C and CD14 markers; and (i) expressionof endothelial CD146, CD202b, and CD31 markers. In a particular aspect,the cell has each of the additional characteristics (f)-(i).

In a seventh embodiment, the present invention is directed to anisolated pathologically-definable circulating tumor cell (CTC), whereinthe cell has one or more of the following characteristics: (a)cancer-like nucleus; (b) expression of one or more of cytokeratin 8, 18and 19, and wherein the cytokeratins have filamentous pattern; and (c)CD45 negative phenotype.

In an eighth embodiment, the present invention is directed to anisolated apoptotic circulating tumor cell (CTC), wherein the cell hasone or more of the following characteristics: (a) cancer-like nucleus;(b) expression of one or more of cytokeratin 8, 18 and 19, and whereinthe cytokeratin is fragmented in the form of spots; and (c) CD45negative phenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the present invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

FIGS. 1A-1D shows circulating tumor cells found in blood of cancerpatients. The merged images are generated by DAPI, CK 8, 18 & 19, EpCAMand CD45 staining.

FIGS. 2A-2D shows apoptotic circulating tumor cells found in blood ofcancer patients. The merged images are generated by DAPI, CK 8, 18 & 19,EpCAM and CD45 staining.

FIG. 3 shows a circulating cancer associated macrophage-like cell (CAML)found in the blood of cancer patients. This merged image is generated byDAPI, CK 8, 18 & 19, EpCAM and CD45 staining.

FIG. 4 shows a circulating cancer associated macrophage-like cell foundin the blood of cancer patients. This merged image is generated by DAPI,CK 8, 18 & 19, EpCAM and CD45 staining.

FIG. 5 shows a circulating cancer associated macrophage-like cell foundin the blood of cancer patients. This merged image is generated by DAPI,CK 8, 18 & 19, EpCAM and CD45 staining.

FIG. 6 shows a circulating cancer associated macrophage-like cell foundin the blood of cancer patients. This merged image is generated by DAPI,CK 8, 18 & 19, EpCAM and CD45 staining.

FIGS. 7A-7I show a gallery of circulating cancer associatedmacrophage-like cells found in the blood of cancer patients. Thesemerged images are generated by DAPI, CK 8, 18 & 19, EpCAM and CD45staining.

FIGS. 8A-8I show the expression of separate CK 8, 18, 19, EpCAM andCD45, and the nucleus, and the merged image of each of the cells in FIG.7. FIGS. 8B, 8C and 8D are from breast cancer patients. FIGS. 8A, 8F and8G are from pancreatic cancer patients. FIGS. 8E, 8H and 8I are fromprostate cancer patients. The merged images are generated by DAPI, CK 8,18 & 19, EpCAM and CD45 staining.

FIG. 9 is whisker plot of cytoplasmic diameters of white blood cells(WBCs), circulating tumor cells (CTCs) and circulating cancer associatedmacrophage-like cells (CAMLs).

FIG. 10 is a comparison of presence of circulating tumor cells (CTCs)captured by CellSearch® and pathologically-definable CTCs isolated byCellSieve™ microfiltration and circulating cancer associatedmacrophage-like cells (CAMLs) isolated at the same time by Cell Sieve™microfiltration.

FIG. 11 is a plot of the number of circulating cancer associatedmacrophage-like cells (CAMLs) found in the circulation of breast,prostate, and pancreatic cancer patients in different stages of cancer.

FIG. 12 is plot of number of CAMLs and CTCs found in the prostate cancerpatient samples.

FIG. 13 is plot of number of CAMLs and CTCs found in the pancreaticcancer patient samples.

FIG. 14 is plot of number of CAMLs and CTCs found in the breast cancerpatient samples.

FIG. 15 is a plot of the number of circulating cancer associatedmacrophage-like cells (CAMLs) found in the circulation of breast,prostate, pancreatic and lung cancer patients in different stages ofcancer.

FIGS. 16A and 16B are plots of number of CAMLs and CTCs found in theprostate cancer patient samples, respectively.

FIGS. 17A and 17B are plot of number of CAMLs and CTCs found in thepancreatic cancer patient samples, respectively.

FIGS. 18A and 18B are plot of number of CAMLs and CTCs found in thebreast cancer patient samples, respectively.

FIG. 19 is plot of number of CAMLs and CTCs found in the lung cancerpatients.

FIG. 20 is plot of number of CAMLs and CTCs found in the colorectalcancer patients.

FIG. 21 is an example of a CAML from a pancreatic cancer patient alsostained for PDX-1.

FIG. 22 is an example of a CAML from a prostate cancer patient alsostained for PSMA.

FIGS. 23A-B show H&E staining of CAMLs. Two representative CAMLs cells(A) and (B) are shown under a light microscope. The blocked arrow is around vacuole located within the cytoplasm of the CAML. Open arrows showthe individual nuclei and subsequent polynuclear nature of the CAMLs.

FIG. 24A is a plot of the number of circulating cancer associatedmacrophage-like cells (CAMLs) in breast cancer patients with notreatment, and hormone treatment or chemotherapy and radiation therapy.

FIG. 24B is a plot of the number of circulating cancer associatedmacrophage-like cells (CAMLs) in pancreatic cancer patients with notreatment, and chemotherapy and radiation therapy.

FIG. 25A shows a CTC associated with a CAML.

FIG. 25B shows a CTC bound to a CAML.

FIG. 25C shows a CTC bound to a CAML with membrane fusing.

FIG. 25D shows a CAML that engulfed a CK positive cell.

FIG. 26A shows examples of CAML from breast cancer patient stained forCD11c, CD14 and TIE-2.

FIG. 26B shows examples of CAML from pancreatic cancer patient stainedfor CD11c, CD14 and TIE-2.

FIG. 26C shows examples of CAML from breast cancer patient stained forCD11c, CD14 and CD146.

FIG. 26D shows examples of CAML from pancreatic cancer patient stainedfor CD11c, CD14 and CD146.

DETAILED DESCRIPTION

The matters defined in the description such as a detailed constructionand elements are nothing but the ones provided to assist in acomprehensive understanding of the invention. Accordingly, those ofordinary skill in the art will recognize that various changes andmodifications of the embodiments described herein can be made withoutdeparting from the scope and spirit of the invention.

Cancer is the most feared illness in the world, affecting allpopulations and ethnicities in all countries. In the United Statesalone, there are more than 10 million cancer patients, with 1.5 millionnew cancer cases and almost 0.6 million deaths per year. Cancer deathworldwide is estimated to be about 8 million annually, of which 3million occur in developed countries where patients have availabletreatment.

Ideally there is a biomarker that can (i) provide early detection of allcarcinomas, especially for at risk groups such as smokers for lungcancer, (ii) confirm other indications of cancer, such as high PSA forprostate cancer, and/or (iii) provide early detection of recurrence ofcancer.

Oncologists need to know how best to treat newly diagnosed cancerpatients. The current testing standard is a tissue biopsy, which is usedto determine the cancer subtype, because therapeutic drugs arefrequently effective only for specific subtypes. The biopsy methodvaries by location, but is invasive and can be risky.

To monitor treatment, oncologists need to know how well the drug isworking for the patient, whether the dose should be adjusted, andwhether the disease is spreading or in remission. The common methods foranswering these questions are x-ray computed tomography (CT) scans andmagnetic resonance imaging (Mills), both of which are expensive.Additionally, these methods cannot provide the necessary informationuntil the tumor size has changed perceptibly.

Ninety percent of cancer patients die from metastasis, not from theprimary tumor. The metastatic process involves tumor cells that breakfree of the primary carcinomas (solid tumors of epithelial cells) andenter the blood stream. These breakaway cancer cells are known ascirculating tumor cells (CTCs). CTCs have the potential to be useful asa tool to determine therapy, monitor treatment, determine recurrence andprovide prognostic information of survival. However, CTCs cannot beconsistently collected from the blood even in stage III and IV cancers.

In this disclosure, a cell type is presented that is consistently foundin the blood of carcinoma patients from stage I-IV. These cells aremacrophage-like cells that contain the same tumor markers as the primarytumor and they are termed circulating Cancer Associated Macrophage-Likecells (CAMLs) herein.

CTCs and CAMLs can be found from the same patient sample at the sametime by size exclusion methods, such as by microfiltration methods.Microfilters can be formed with pores big enough to let all red bloodcells and majority of white blood cells through and retain larger cellssuch as CTCs and CAMLs. Size exclusion methods have also beenimplemented by microfluidic chips.

CAMLs have many clinical utilities when used alone. Furthermore, CAMLscan be combined with other markers such as CTCs, free DNA in blood andfree proteins in blood to further improve sensitivity and specificity ofa diagnosis. This is especially true for CAMLS and CTCs because they canbe isolated and identified at the same time.

Circulating Tumor Cells

The CTCs express a number of cytokeratins (CKs). CK 8, 18, & 19 are themost commonly used in diagnostics, but surveying need not be limited tothese three. The surface of solid tumor CTCs usually express epithelialcell adhesion molecule (EpCAM). However, this expression is not uniformor consistent. CTCs should not express any CD45, because it is a whiteblood cell marker. In assays to identify tumor associated cells, such asCTCs and CAMLs, it is sufficient to use antibody against CK 8, 18, or19, or antibody against CD45 or DAPI. Combining the presence of stainingwith morphology, pathologically-definable CTCs, apoptotic CTCs and CAMLscan be identified.

FIGS. 1A-1D show four examples of pathologically-definable CTCs. Apathologically-definable CTC is identified by the followingcharacteristics:

-   -   They have a “cancer-like” nucleus stained by DAPI. The exception        is when the cell is in division; the nucleus is condensed.    -   They express at least CK 8, 18 and 19. The cytokeratins have a        filamentous pattern.    -   They lack CD45 expression. To avoid missing low expressing CD45        cells, long exposure is used during image acquisition.

A pathologically-definable CTC of the present invention thus includesthose CTCs having one, two or three of the following characteristics:(a) cancer-like nucleus; (b) expression of one or more of cytokeratin 8,18 and 19, and wherein the cytokeratins have filamentous pattern; and(c) CD45 negative phenotype.

FIG. 1A shows a pathologically-definable prostate cancer CTC expressingwell-defined EpCAM and FIG. 1B shows a pathologically-definable prostatecancer CTC expressing very low or no EpCAM. FIG. 1C shows apathologically-definable breast cancer CTC expressing well-defined EpCAMand FIG. 1D shows a pathologically-definable breast cancer CTCexpressing very low or no EpCAM.

FIGS. 2A-2D show examples of apoptotic CTCs. An apoptotic CTC isidentified by the following characteristics:

-   -   They have a cancerous nucleus.    -   They express at least CK 8, 18 and 19; the cytokeratins are not        filamented, but appear fragmented in the form of spots.    -   They do not express CD45.

An apoptotic CTC of the present invention thus includes those CTCshaving one, two or three of the following characteristics: (a)cancer-like nucleus; (b) expression of one or more of cytokeratin 8, 18and 19, and wherein the cytokeratin is fragmented in the form of spots;and (c) CD45 negative phenotype.

FIGS. 2A and 2B show apoptotic breast cancer CTCs expressing very low orno EpCAM at early and mid stages of apoptosis, respectively. FIGS. 2Cand 2D show prostate cancer CTCs at mid stage of apoptosis expressinghigh and low EpCAM, respectively.

Circulating Cancer Associated Macrophage-Like Cells (CAMLs)

In the same patient samples, another type of cell was identified. Thiscell type has been termed a CAML. CAMLs have the followingcharacteristics:

-   -   CAMLs have a large atypical nucleus; multiple individual nuclei        can be found in CAMLs, though enlarged fused nucleoli        approximately 14 μm to approximately 65 μm are common.    -   CAMLs may express at least CK 8, 18 or 19, and the CK is        diffused, or associated with vacuoles and/or ingested material.        CK is nearly uniform throughout the whole cell.    -   CAMLs are most of the time CD45 positive.    -   CAMLs are large, approximately 20 micron to approximately 300        micron in size.    -   CAMLs come in five distinct morphological shapes (spindle,        tadpole, round, oblong, or amorphous).        Further analysis of CAMLs shows they also have the follow        characteristics:    -   If CAML express EpCAM, EpCAM is diffused, or associated with        vacuoles and/or ingested material, and nearly uniform throughout        the whole cell, but not all CAML express EpCAM, because some        tumors express very low or no EpCAM.    -   CAML express markers associated with the markers of the tumor        origin; e.g., if the tumor is of prostate cancer origin and        expresses PSMA, then CAML from this patient also expresses PSMA.        Another example, if the primary tumor is of pancreatic origin        and expresses PDX-1, then CAML from this patient also expresses        PDX-1.    -   CAMLs express monocytic markers (e.g. CD11c, CD14) and        endothelial markers (e.g. CD146, CD202b, CD31). CAMLs also have        the ability to bind Fc fragments.

CAMLs of the present invention thus includes those CAMLs having one,two, three, four or five of the following characteristics: (a) largeatypical nucleus having a size of about 14-64 μm; (b) expression of oneor more of cytokeratin 8, 18 and 19, wherein the cytokeratin isdiffused, or associated with vacuoles and/or ingested material; (c) cellsize ranging from about 20 micron to about 300 microns; (d)morphological shape selected from the group consisting of spindle,tadpole, round, oblong, and amorphous; and (e) CD45 positive phenotype.CAMLs of the present invention also include those CAMLs having one, two,three or four of the following additional characteristics: (f)expression of diffuse EpCAM with nearly uniform distribution; (g)expression of one or more markers of a primary tumor; (h) expression ofmonocytic CD11C and CD14 markers; and (i) expression of endothelialCD146, CD202b, and CD31 markers. In a particular aspect, CAMLs of thepresent invention have each of the additional characteristics (f)-(i).

Images of CAMLs from a breast cancer patient are shown in FIGS. 3-5 andCAMLs from a prostate cancer patient are shown in FIG. 6. FIG. 7contains a collage of CAMLs showing the five different CAML morphologiesand signal variation from separate prostate, breast and pancreaticpatient samples: (7A) pancreatic, (7B) breast, (7C) breast, (7D) breast(7E) prostate, (7F) pancreatic, (7G) pancreatic, (7H) prostate, and (7I)prostate. Examples of morphology variants are as follows: amorphous(7A), oblong (7B and 7G), spindle (3, 5, 6, 7C, 7F and 7I), round (7D)and tadpole (4, 7E & 7H). Color differences occur from varying degreesof protein expression from antibody reaction to EpCAM, cytokeratin andCD45. The expression of markers of each of the CAMLs in FIGS. 7A-I areshown in detail in FIGS. 8A-I.

FIG. 9 shows the whisker plot of cytoplasmic diameters of white bloodcells (WBC), CTCs and CAMLs. The median size differences, and ranges,between WBC, CTCs and CAMLs captured on the microfilter from prostate,breast, and pancreatic (n=25 white blood cells, n=25 CAMLs and n=25CTCs). Diameters were measured using the distance between the twolongest points on the cell using Zen 2011 measurement software. Themedian value of white blood cells was 12.4 μm, CTCs was 18.8 μm andCAMLs was 43.5 μm.

Diagnostic Methods of Using CAMLs and CTCs

As suggested above, the unique characteristics of the CAMLs and CTCsdescribed herein make them well-suited for use in clinical methodologyincluding methods of screening and diagnosis diseases such as cancer,and in the monitoring of disease progression.

The invention is thus directed, in a first embodiment, to methods ofscreening a subject for cancer, comprising detecting circulating cancerassociated macrophage-like cells (CAMLs) in a biological sample from asubject. In particular aspects, when CAMLs are detected in thebiological sample, the subject is identified as potentially having acarcinoma or solid tumor. In other aspects, when CAMLs are detected inthe biological sample, the subject is identified as having a carcinomaor solid tumor. In certain aspects, the methods encompassed by thisembodiment also include detecting circulating tumor cells (CTCs) in thebiological sample. In particular aspects of this first embodiment, thesubject is a subject suspected of having cancer.

In a second embodiment, the invention is directed to methods fordiagnosing cancer in a subject, comprising detecting CAMLs in abiological sample from a subject, wherein when CAMLs are detected in thebiological sample, the subject is diagnosed with cancer. In certainaspects, the methods encompassed by this embodiment also includedetecting CTCs in the biological sample, wherein when CAMLs and CTCs aredetected in the biological sample, the subject is diagnosed with cancer.

In a third embodiment, the invention is directed to methods fordetecting recurrence of cancer in a subject, comprising detecting CAMLsin a biological sample from a subject previously treated for cancer,wherein when CAMLs are detected in the biological sample, recurrence ofcancer is detected. In certain aspects, the methods encompassed by thisembodiment also include detecting CTCs in the biological sample, whereinwhen CAMLs and CTCs are detected in the biological sample, recurrence ofcancer is detected.

In a fourth embodiment, the invention is directed to methods forconfirming a diagnosis of cancer in a subject, comprising detectingCAMLs in a biological sample from a subject diagnosed with cancer,wherein when CAMLs are detected in the biological sample, a diagnosis ofcancer is confirmed in the subject. In certain aspects, the methodsencompassed by this embodiment also include detecting CTCs in thebiological sample, wherein when CAMLs and CTCs are detected in thebiological sample, a diagnosis of cancer is confirmed in the subject. Inparticular aspects, the initial cancer diagnosis is via mammography, PSAtest, or presence of CAl25. In a particular aspect, the subject issuspected of having cancer.

In each of these embodiments, CAMLs are detected using appropriate meanswhich include, but are not limited to, one or more of size exclusionmethodology, red blood cell lysis, FICOLL, a microfluidic chip, and flowcytometry, or a combination thereof. When size exclusion methodology isutilized, it may comprise the use of a microfilter. Suitablemicrofilters can have a variety of pore sizes and shapes. Microfiltershaving pores of about 7-8 microns in size are acceptable, and includeround and rectangular pore shapes. Microfilters having round pores ofabout 7-8 microns in size are especially optimal when polymericmicrofilters are used. In a preferred aspect, the microfilter hasprecision pore geometry and uniform pore distribution.

Alternatively, CAMLs may be detected using a microfluidic chip based onmeans that include, but are not limited to, physical size-based sorting,hydrodynamic size-based sorting, grouping, trapping, immunocapture,concentrating large cells, or eliminating small cells based on size.

In each of these embodiments, CTCs may also be detected along withCAMLs. Such detection may be simultaneously or sequential detection, andcan utilize the same or different means. For example, simultaneousdetection using a microfilter having a pore size that selects for bothcell types may be used. Suitable microfilters can have a variety of poresizes and shapes. Microfilters having pores of about 7-8 microns in sizeare acceptable, and include round and rectangular pore shapes.Microfilters having round pores of about 7-8 microns in size areespecially optimal when polymeric microfilters are used. In a preferredaspect, the microfilter has precision pore geometry and uniform poredistribution.

The methods provided in these embodiments may be practiced using anybiological sample suspected of containing CTCs and/or CAMLs. Suitablebiological samples include, but are not limited to, one or more selectedfrom the group consisting of peripheral blood, blood, lymph nodes, bonemarrow, cerebral spinal fluid, tissue, and urine. In a preferred aspect,the biological sample is peripheral blood. In other aspects, the bloodis antecubital-vein blood, inferior-vena-cava blood or jugular-veinblood.

The skilled artisan will appreciate that the methods provided in theseembodiments are not limited to particular forms or types of cancer andthat they may be practiced in association with a wide variety ofcancers. Exemplary cancers include, but are not limited to, solidtumors, Stage I cancer, Stage II cancer, Stage III cancer, Stage IVcancer, epithelial cell cancer, breast cancer, prostate cancer, lungcancer, pancreatic cancer, and colorectal cancer.

Monitoring Treatment Efficacy Using CAMLs and CTCs

As suggested above, the unique characteristics of the CAMLs and CTCsdescribed herein also make them well-suited for use in monitoring theeffectiveness of disease treatments.

The present invention is thus directed, in a fifth embodiment, tomethods for monitoring efficacy of a cancer treatment, comprising:

(a) determining the number of CAMLs in a biological sample from asubject before cancer treatment, and

(b) comparing the number of CAMLs determined in (a) to a number of CAMLsdetermined from a similar biological sample from the same subject at oneor more time points after treatment.

Given the prognostic capabilities associated with CTCs, the methods ofthis embodiment may further include the following additional steps:

(c) determining the number of CTCs in the biological sample of (a), and

(d) comparing the number of CTCs determined in (c) to a number of CTCsdetermined from the biological sample of (b).

The skilled artisan will understand that a change in the number of CAMLsand/or CTCs will be an indication of treatment efficacy, where thechange may be an increase or a decrease in the number of CAMLs and/orCTCs.

The particular cancer treatment is not limited by this method, but willgenerally comprise chemotherapy and/or radiation therapy.

These methods can be practiced by determining number of CAMLs using oneor more means selected from the group consisting of size exclusionmethodology, red blood cell lysis, FICOLL, a microfluidic chip, and flowcytometry, or a combination thereof. When size exclusion methodology isused, it may comprise use of a microfilter. Suitable microfilters canhave a variety of pore sizes and shapes. Microfilters having pores ofabout 7-8 microns in size are acceptable, and include round andrectangular pore shapes. Microfilters having round pores of about 7-8microns in size are especially optimal when polymeric microfilters areused. In a preferred aspect, the microfilter has precision pore geometryand uniform pore distribution. In a particular aspect, the number ofCAMLs is determined using a microfluidic chip based on means thatinclude, but are not limited to, physical size-based sorting,hydrodynamic size-based sorting, grouping, trapping, immunocapture,concentrating large cells, or eliminating small cells based on size. Inanother particular aspect, the number of CAMLs is determined using aCellSieve™ low-pressure microfiltration assay.

When both are monitored, the numbers of CAMLs and CTCs may be determinedsequential or simultaneously using the same means or different means.For example, simultaneous detection using a microfilter having a poresize that selects for both cell types may be used. Suitable microfilterscan have a variety of pore sizes and shapes. Microfilters having poresof about 7-8 microns in size are acceptable, and include round andrectangular pore shapes. Microfilters having round pores of about 7-8microns in size are especially optimal when polymeric microfilters areused. In a preferred aspect, the microfilter has precision pore geometryand uniform pore distribution.

The methods provided in this embodiment may be practiced using anybiological sample suspected of containing CTCs and/or CAMLs. Suitablebiological samples include, but are not limited to, one or more selectedfrom the group consisting of peripheral blood, blood, lymph nodes, bonemarrow, cerebral spinal fluid, tissue, and urine. In a preferred aspect,the biological sample is peripheral blood. In other aspects, the bloodis antecubital-vein blood, inferior-vena-cava blood or jugular-veinblood

The skilled artisan will appreciate that the methods provided in thisembodiment are not limited to particular forms or types of cancer andthat they may be practiced in association with a wide variety ofcancers. Exemplary cancers include, but are not limited to, solidtumors, Stage I cancer, Stage II cancer, Stage III cancer, Stage IVcancer, epithelial cell cancer, breast cancer, prostate cancer, lungcancer, pancreatic cancer, and colorectal cancer.

Discussions of the Biology and Clinical Results of CAMLs

Tumor-associated macrophages (TAMs) are specialized differentiatedmacrophages found within most tumors, which can be used as prognosticindicators of either tumor invasiveness or tumor suppression. TAMs,recruited to the stroma from circulating monocytes, are required fortumor cell intravasation, migration, extravasation, and angiogenesis.Tumors attract monocytes via chemoattractants (MCP-1, CCL-2). In turnTAMs secrete cytokines, chemokines and growth factors (e.g. MMP-1,CXCL12) which stimulate tumor cells with the potential to becomecirculating tumor cells (CTCs). TAMs and tumor cells then migrate viathe lymphatic system, or intravasate across intra-tumor capillarybarriers into the peripheral circulation.

Pathological evidence detailing the initial dissemination steps of CTCsvia a metastatic cascade remains inconclusive. Typically, a cancer celldissemination cascade requires 3 steps: CTC separation from the tumor,movement away from the parent mass, and migration into the circulatorysystem. Though various theories explain select aspects of this cascade(endothelial progenitor cells, cancer mesenchymal stem cells, hybridcancer cells, etc.), none completely explain all three processes.Recently, in vivo studies have shown that circulating monocytic cellsare intricately involved in tumor cell invasiveness, motility, andmetastatic potential. Interactions between myeloid lineage cells andtumor cells have been documented in patients and modeled in micesuggesting a pathway for intravasation, but the mechanism for the finaldissemination step is yet to be established.

Here we describe the existence of CAMLs, a highly differentiated giantcirculating (macrophage-like) cell isolated from the peripheral blood ofbreast, prostate, and pancreatic cancer patients, which we hypothesizecould be a disseminated TAM (DTAM). We isolated this cell type usingCellSieve™ microfilter (Creatv MicroTech) with precision pore dimensionsand uniform distribution. CellSieve™ microfiltration of 7.5 ml of wholeperipheral blood are performed under low pressure without damagingcells, allowing for histological identification of cellular morphology.We define this giant cell as a circulating Cancer AssociatedMacrophage-Like cell (CAML), as it exhibits a CD14+ expression, vacuolesof phagocytosed material, and found exclusively in cancer patients(FIGS. 3-7, and Table 1). We propose that this cell population, notfound in healthy individuals, could serve as a robust cellular biomarkerof a previously undefined innate immune response to canceraggressiveness, and monitor chemotherapy- and radiation therapy-inducedresponses. Observations of these giant cells interacting with CTCs whilein circulation supports evidence that a patient's innate immune responsehas an observable effect on the migration of CTCs. The TIE-2 positivemarkers expressed by these macrophages suggest a role of CAML ascellular initiators of neovascularization within tumor metastases. Wehave uncovered supporting in vivo evidence that CAMLs may play anassociated role in the migration of CTCs in circulation.

Giant fused macrophages are a poorly understood cell found in amultitude of tissues. They are hybrids of multinucleated cellsoriginating from myeloid lineage involved in numerous physiological andpathological processes, including phagocytosis of foreign and necrotictissue, tissue reabsorption, and inflammation. We find that CAMLs aregiant cells presenting with enlarged nuclei, CD45+ and exhibit diffusedcytoplasmic staining characteristic of epithelial cells: cytokeratin 8,18, 19, and epithelial cell adhesion molecule (EpCAM) (FIGS. 3-8).Multiple individual nuclei can be found in CAMLs, though large fusednucleoli (14-64 μm diameter) are common (FIGS. 3-8). CAML cytoplasm,defined by a cytokeratin border, range from 21-300 um in length and isfound on the filter with five distinct morphological phenotypes(spindle, tadpole, round, oblong, or amorphous) (FIGS. 3-8).

Although identification of these cells is straightforward due to theirextreme size, large nuclear profile, and cytoplasmic signature, theyhave highly heterogeneous phenotypes. The expression of cytokeratin,EpCAM and CD45 all vary from lack of expression to very intenseexpression (FIGS. 8A-8H). This heterogeneity is further indicated in thefive cell structures, the size ranges, and the various nuclear profiles(FIGS. 8A-8H). High marker expression heterogeneity implies CAMLsrepresent either different stages along pathways of differentiation orare the product of nonspecific cellular engulfment with varying celltypes. This is unsurprising as macrophages are a highly plastic celltype capable of differentiating into numerous cell phenotypes.

Recognizing that CAMLs could serve as an independent prognosticindicator of cancer progression, similar to CTC enumeration, we comparedthe number of CAMLs and CTCs using the CellSieve™ low-pressuremicrofiltration assay (Creatv MicroTech, Inc) and the CellSearch®Circulating Tumor Cell test (Vendex, LLC). CTCs have been a challenge toisolate due to their rarity and limited occurrence (10-50%) in cancerpatients with metastatic disease. TAM enumeration and phenotyping couldhave prognostic utility, but currently lacks sequential testing,tracking primary to metastatic progression, as this would requirenumerous invasive tumor biopsies. Enumeration of CTCs enriched byCellSearch® and CellSieve™ systems were directly compared to theenumeration of CAMLs isolated by CellSieve™ using blood from 29 cancerpatients (FIG. 10). While CellSearch® uses EpCAM antibodies to enrichCTCs, CellSieve™ uses size exclusion. Both technologies phenotypicallyidentify CTCs utilizing an antibody panel of anti-cytokeratin 8, 18, and19, DAPI nuclear stain and absence of anti-CD45. Employing a CTC countof ≥1, the sensitivity of the CellSieve™ system was 72% [21 of 29patients; breast=15/21 (71%), prostate=6/8 (75%)], while that of theCellSearch® system was 58% [15 of 29 patients; breast=9/21 (43%),prostate=6/8 (75%)]. CAML capture was positive in 97% (28 of 29) ofsamples tested (FIG. 10). Interestingly, the only CAML negative samplewas a breast cancer patient being treated with bisphosphonates, a classof drugs, which inhibit formation of osteoclasts, a giant myeloid cellof the bone composed of fused cells. Thus, as the specificity of CAMLsis 100% for the samples tested, presence of CAML may provide a methodfor non-invasive sequential testing for use as a prognostic indicator ofmetastatic disease for a broad range of cancer patients.

To assess the sensitivity and specificity of CAMLs for applicability asa prognostic indication of metastatic disease, we examined samples fromearly to late stage cancer patients, and healthy subjects. Samples from76 patients were run on CellSieve™ microfilters. The stage distributionincluded, Stage I (n=8), Stage II (n=9), Stage III (n=13), Stage IV(n=24), unknown stage (n=22) across three cancers: breast (n=29),pancreatic (n=27), and prostate (n=20) (Table S1). We included newlydiagnosed and untreated patients (n=36) as well as patients undergoingnon-surgical therapies (n=40) (Table 1). The study included healthysubjects (n=30), including two with benign disease, one fibroadenoma andone basal cell carcinoma. No CAMLs were found in this control group(Table 1). CAMLs were found in 97% (36 of 37) cancer patients with StageIII/IV cancer, 77% (13 of 17) of Stage I/II, and 93% (71 of 76) of allpatients regardless of cancer type (FIG. 11). CAMLs were found in 85%(prostate) (FIG. 12), 93% (pancreatic) (FIG. 13), and 97% (breast) (FIG.14) of the patients. CAMLs in prostate cancer samples were slightlylower in number, possibly due to the fact that 6 of 20 prostate patientswere stage I. Although CTC analysis on microfilters provided an overallpositivity of 54% across all patients tested, CAMLs provided 93%sensitivity across the same cohort (FIGS. 12-14 and Table 1). Thoughfurther study of patients with various illnesses must be assessed, thesefindings demonstrate that the presence CMALs could provide a robust andwidely applicable assessment of cancer status.

TABLE 1 Summary of healthy subjects, CAML patient data and CTC patientdata separated by stage, cancer type and therapy type. CTCs are found ina small percent of Stage I and II patients, while CAMLs are found inmany stage I-IV patients. CTC numbers between the patients is highlyvariable with a standard deviation (SD) of >300%. CAMLs have a muchlower SD, 110%, and whose numbers seem to correlate with stage andtreatment. No. of CAML Mean CAML ± Median CAML CTC Mean CTC ± Median CTCSubject patients positive (%) SD CAML range positive (%) SD CTC rangeHealthy Normals 28 0 0 ± 0 0 0-0  0 0 ± 0 0 0-0  Nonmalignant 2 0 0 ± 00 0-0  0 0 ± 0 0 0-0  Stage I 8 63 10.8 ± 20.9 3 0-61 38  4.6 ± 11.9 00-34  Stage II 9 89 8.0 ± 9.6 3 0-28 22 1.6 ± 3.1 0 0-28  Stage III 13100 15.8 ± 12.5 9.5 2-43 62 13.3 ± 30.2 3.5 0-108 Stage IV 24 96 21.3 ±23.8 8 0-82 75  57.2 ± 172.4 3 0-682 Unknown 22 95 6.2 ± 4.5 5.5 0-20 45 4.8 ± 17.5 0 Cancer Type 76 93 13.4 ± 17.4 6 0-82 54  22.5 ± 100.3 10-682 Breast (IBC) 14 93 24.9 ± 24.0 23.5 0-82 71 11.4 ± 28.1 3 0-108Breast (IDC) 8 100 15.9 ± 16.1 10.5 1-41 100  9.0 ± 17.0 3.5 Jan-51Breast 7 100 15.0 ± 14.2 8 2-32 71 106.3 ± 254.2 5 0-682 (unknown)Prostate 20 85  9.0 ± 14.3 3 0-64 35 3.3 ± 7.9 0 0-34  Pancreatic 27 93 9.5 ± 14.5 5 0-72 41  24.8 ± 106.5 0 0-541 Therapy Type 76 No treatment36 86 4.4 ± 4.0 3.5 0-19 42 20.2 ± 92.0 0 0-541 Hormone 13 92 11.8 ±16.7 8 0-64 69 6.2 ± 9.6 3 0-34  Chemotherapy 24 100 26.2 ± 21.9 27 2-8258  8.9 ± 22.6 2 0-108 Unknown 3 100 26.3 ± 11.6 25 13-34  100 229.3 ±392.0 4 2-682

As more samples were collected, additional CAML data was obtained. FIG.15 presents CAML counts for 122 patients with staging information forbreast, pancreatic, prostate and non-small cell lung cancer (NSCLC). ForStage I, one pancreatic cancer patient and six prostate cancer patientsdid not have any CAMLs. For Stage II, one pancreatic cancer patient andone prostate cancer patient did not have any CAMLs. For Stage IV, onebreast cancer patient, one lung cancer patient and three pancreaticcancer patients did not have any CAMLs.

FIGS. 16A and 16B present numbers of CAMLs and CTCs from prostate cancerpatient data obtained to date, respectively. Staging information was notknown for all patients. The percentage of prostate cancer patients withCAMLs was 81% (30/37) (FIG. 16A), where 13 are known as Stage I. Thepercentage of prostate cancer patients with pathologically-definableCTCs was 32% (12/37) (FIG. 16B).

FIGS. 17A and 17B present numbers of CAMLs and CTCs from pancreaticcancer patient data obtained to date, respectively. Staging informationwas not known for all patients. The percentage of pancreatic cancerpatients with CAMLs was 93% (71/76) (FIG. 17A), where 11 were known asStage I. Two samples had pathologically-definable CTCs larger than 80.Samples No. 13 and 53 had 83 and 541 pathologically-definable CTCs,respectively. The percentage of prostate cancer patients withpathologically-definable CTCs was 23% (18/76) (FIG. 17B).

FIGS. 18A and 18B present numbers of CAMLs and CTCs from breast cancerpatient data obtained to date, respectively. Staging information was notknown for all patients. The percentage of breast cancer patients withCAMLs was 97% (36/37) (FIG. 18A), where only one was known as Stage I.One Stage IV patient did not have any CAML because she was taking thebisphosphonates. Four samples had pathologically-definable CTCs largerthan 110. Samples No. 17, 27 and 30 had 978, 682 and 707pathologically-definable CTCs, respectively. The percentage of breastcancer patients with pathologically-definable CTCs was 84% (31/37) (FIG.18B).

FIG. 19 presents numbers of CAMLs and CTCs from NSCLC patient dataobtained to date. Staging information was not known for all patients.The percentage of NSCLC patients with CAMLs was 88% (7/8). Thepercentage of NSCLC patients with pathologically-definable CTCs was 38%(3/8).

FIG. 20 presents numbers of CAMLs and CTCs from colorectal cancerpatient data obtained to date. All the patients were Stage IV. Thepercentage of colorectal cancer patients with CAMLs was 100% (4/4) forthis small sample size. The percentage of colorectal cancer patientswith pathologically-definable CTCs was 25% (1/4).

In all these data, the CAMLs were found in much larger number of thepatients than CTCs.

Though further study of patients with various illnesses must beassessed, these findings demonstrate that the presence CAMLs provides arobust and widely applicable assessment of cancer status for carcinomas.

We propose that CAML represents a specialized TAM initiating at the siteof tumor and disseminating into circulation. This would be consistentwith previous publications characterizing TAMs at the primary tumorsite, as well as the observations that CAMLs appear CD14+, have engulfedepithelial tissue, and occur exclusively in cancer patients. Tounderstand the origin of CAMLs, we looked at whether they arise fromcirculating monocytes or directly from DTAMs. As TAMs and monocyteswould both present with similar protein markers, we assessed thepresence of engulfed organ-specific markers, Pancreatic DuodenalHomeobox-1 (PDX-1) for pancreatic patients or Prostate-Specific MembraneAntigen (PSMA) for prostate patients. PDX-1 is a differentiation anddevelopment marker found in adult endocrine organs, namely pancreaticcells. PSMA is a membrane glycoprotein, which is highly expressed inprostate cells. After isolating and enumerating CAMLs, we re-stainedpre-identified CAML samples with PSMA or PDX-1. A PDX-1 positivereaction was seen in all CAMLs from pancreatic samples (FIG. 21), whilePSMA was found in all CAMLs from prostate samples (FIG. 22). Whilepossible that cellular fusion or ingestion of debris occurred away fromthe tumor site, the high concentration of markers coupled with thescarcity of tumor debris in circulation make this unlikely. Instead, weinterpret this suite of biomarker staining as evidence that CAMLs are asubtype of DTAMs, and that staining occurred from phagocytosed necroticdebris, or engulfed neoplastic cells from the tumor site.

TABLE 2 Cell markers used to analyze the CAMLs. TIE-2 Cytokeratin EpCAMCD45 Cell Type PDX-1 PSMA CD11c CD14 CD146 (C202b) 8, 18 & 19 (CD326)(LCA) CCAMLC   +(*)   +(‡) + +/− +/− +/− + + +/− Breast CTC − − − − −− + +/− − Prostate CTC − + − − − − + +/− − Pancreas CTC + − − − − − ++/− − Epithelial Cell − − − − − − + + − Monocyte − − + +   −(♦)   −(♦) −− + Endothelial Cell − − − − + + + − − White Blood Cell − −   −(∘)  −(∘) − − − − + (+) Positive in the majority of cell types; (−)negative in the majority of cell types; (+/−) cell populationsheterogenous for this marker and may be positive or negative; (*)foundonly in cells from pancreatic cancer patients; (‡)found only in cellsfrom prostate cancer patients; (♦)a small subset population on monocytesare positive for this marker; (∘)monocytes are a subpopulation of whiteblood cells and will express these markers.

FIG. 23 shows H & E Staining of CAMLs. Samples from breast cancerpatients were identified by fluorescent DAPI and cytokeratin stains.Filters were then re-stained by Hematoxylin and by Eosin Y. Tworepresentative CAMLs cells are shown under a light microscope. Theblocked arrow is a round vacuole located within the cytoplasm of theCAML. Open arrows show the individual nuclei and subsequest polynuclearnature of the CAMLs (A) An oval shaped CAMLs that has 3 visible nucleiand a vacuole (B) Round shaped CAML that has 5 visible nuclei.

To further test whether CAMLs are a DTAM subtype, we compared therapyregimes and temporal changes in the number of CAMLs in relation tocancer progression and stability. We hypothesized that if CAMLs areassociated with phagocytosis of cellular debris due to cytotoxicty atthe tumor site, i.e. derived from TAMs, then patients notchemo-responsive or undergoing only hormone therapy would not produceadditional cellular debris and thus would have no change in CAMLnumbers. Conversely, patients that are responsive to chemotherapy wouldhave an increase in CAMLs. Analysis of therapeutic regimes showed thatonly chemotherapy, not hormonal nor non-therapy was associated with anincrease in CAML levels in breast cancer patients (FIG. 24A) andpancreatic cancer patients (FIG. 24B). For Pancreatic cancer patients,CAMLs for chemotherapy average is 16.4 CAMLs, and for no therapy is 4.1.We find that non-treated and hormone treated patients, who should showlittle change in tumor size, have low numbers of CAMLs <0.4-0.6/mL blood(FIGS. 24A-B). Conversely, if a chemotherapeutic regime was in use, CAMLnumbers increased to 3.9/mL. These data suggest that CAMLs may provide asensitive representation of phagocytosis at the tumor site that couldquantify a cell-specific immune response to the extent of cellulardebris caused by chemotherapy.

CTCs originate at a tumor site, circulate in peripheral blood, and havethe ability to seed metastatic sites. However, the pathway for CTCdetachment and invasion into the circulatory system is a complexprocess. We analyzed CAMLs isolated from patient samples for evidence ofa CTC/CAML interaction. CTCs were found bound to CAMLs in 3 of 72patients, all in metastatic disease (FIGS. 25A-25C). In addition, threeinstances of CAMLs were found with engulfed cells that appeared to havea neoplastic/CTC phenotype (FIG. 25D). Representing 7% of patients, theobserved interaction of a dual pair CTC and CAML is indicative of twopossibilities. First, these cells attached while in circulation,implying that CAMLs are an active immune response to cancer cells inblood. Second, these cells bind at the primary tumor and disseminatedtogether into circulation, implying a similar pathway of intravasation.In either case, this pairing of cells suggests that CAMLs may play arole in the immune response to cancer cell migration in the peripheralblood of cancer patients.

Circulating monocytes have the ability to enter any tissue compartmentof the body, including lymph nodes, bone marrow, most organs, and evencross the blood brain barrier. Angiogenic Endothelial Progenitor Cells(EPCs) with neovascular potential are capable of being derived frommacrophages, and TIE-2+ (CD202b) macrophages are intricately involved intumor vascularization. Recent in vitro and mouse in vivo experimentshave shown that EPCs derived from CD14+/CD11c+ monocytes differentiateinto CD146+/TIE-2+ endothelial cells capable of pro-angiogenic activity.As CAMLs presented with an EPC-like spindle phenotype, we analyzed CAMLsfor evidence of this pathway. After identifying CAML positive samples,we stained CAMLs with panels of monocytic markers, CD11c and CD14, aswell as angiogenic endothelial markers, CD146 or TIE-2 (FIGS. 26A-26D).We observed CAMLs positive for both monocytic and endothelial markers.The monocytic marker CD11c was the most reactive, found in all CAMLsstained, while a secondary monocytic marker CD14 was the least reactive,at times negative. The pro-angiogenic marker (TIE-2) and endothelialmarker (CD 146) stained positive in CAMLs, but staining intensity wasintermittent. The endothelial/monocytic overlap findings are notsurprising, as circulating monocytes have high morphological and markerheterogeneity. Even today there is great debate over the mononuclearphagocytic system for classification as endothelial cells, bonemarrow-derived cells and monocytic cell express overlapping markers andhave similar developmental pathways. Nevertheless, the presence ofCD146, or TIE-2, on CD14 positive cells indicates a specializedpro-angiogenic macrophage capable of neovascular potential.

Although many studies have focused on dissemination of CTCs, TAMs may beinvolved in seeding, proliferation and neovascularization of metastases.While previous studies provide evidence for vascular infiltration oftumor cells through macrophage assistance, our results now also supplyevidence of macrophage interaction with tumor cells in the circulation.We hypothesis that CAMLs originating at the primary tumor disseminateinto the circulation. We also show that CAMLs bind to and migratethrough the circulation attached to CTCs, possibly disseminating inconjunction. Finally, we describe pro-angiogenic TIE-2/CD146-expressingCAMLs, indicating the ability to neovascularize a metastaticmicroenvironment. These data provide clinical evidence thatpro-angiogenic cells migrate bound to CTCs, suggesting a link betweenintravasation, migration and extravasation of CTCs.

Capture of CAMLS and CTCs

Cells larger and/or less flexible than other cells present in a bodilyfluid may be collected by filtering the bodily fluid. For example,targeted cells indicative of a condition may be collected by passing abodily fluid through a filter having openings that are too small for thetarget cells to pass through, but large enough for other cells to passthrough. Once collected, any number of analyses of the target cells maybe performed. Such analyses may include, for example, identifying,counting, characterizing expressions of markers, obtaining molecularanalysis, and/or culturing the collected cells.

CAMLs, pathologically-definable CTCs, and apoptotic CTCs are larger thanred blood cells and most white blood cells. Using a precisionmicrofilter that has precision pore size and pore distribution has beenshown to provide high capture efficiency and low standard of deviation.CellSieve™ microfilters (Creatv MicroTech) are one example of precisionmicrofilters. CellSieve™ microfilters are transparent and nonfluorescentmaking them ideal for microscope imaging analysis. Pore sizes of 7-8microns eliminated all the red blood cells and 99.99% of the white bloodcells. Methods to fabricate microfilters producing uniform pore size anddistribution are described in WO 2011/139445, and PCT/US12/66390, bothof which is incorporated herein by reference in their entireties.Microfilters made by a track etch method have randomly located poresthat can overlap resulting in effectively large pores. They might losesome CAMLs and CTCs.

Many other methods exist for captures of CTCs. Some can also be adoptedto capture CAMLs. They generally break-down into the followingcategories:

-   -   Since CAMLs are large compared with majority of blood cells, any        size based method is suitable for capturing CAMLs. Microfilters        are ideal for capture of CAMLs and CTCs. Microfluidic chips        technologies that sort, select, group, trapping, concentrates        large cells or eliminate small cells by size are also suitable.    -   Immunocapture use ferrofluids, magnetic beads, microfluidic        chips, etc, coated with antibody for selection of CAMLs, or        elimination of other cells.    -   Red blood cell lysis can also be used for collecting CAMLs. The        resultant sample volume requires plating on multiple glass        slides.    -   FICOLL.    -   Flow cytometry.    -   A variety of microfluidic chip utilizing a variety of biological        and physical principles.        Summary of Clinical Utilities

These results support the idea that CAMLs provide a robust indicator ofcancer presence.

The sensitivity and specificity of the utility of CAMLs can be furtherimproved in combination with simultaneous detection of CTCs.

Cancer screening is a strategy used in a population to identify anunrecognised disease in individuals without signs or symptoms, withpre-symptomatic or unrecognised symptomatic disease. As such, screeningtests are somewhat unique in that they are performed on personsapparently in good health. A screening test is not a diagnostic test.Diagnostic testing is a procedure performed to confirm, or determine thepresence of disease in an individual suspected of having the disease.

CAMLs can be used as a cancer diagnostic to provide additionalnon-invasive diagnostics to confirm other screening techniques, such asmammography, PSA and CAl25.

Since CAMLs can be found in stage I and II of cancer, CAMLs can be usedas screening for early detection of carcinomas of epithelial originespecially for high risk patients for lung, pancreatic, colorectal andother cancers that does not have early detection methods. Specificity ofthe type of cancer can be determined by staining for various cancer sitespecific markers. Some examples are (i) use antibody against PSMA tospecifically identifying prostate cancer, (ii) use antibody againstPDX-1 to specifically identifying lung cancer, (iii) antibody againstCAl25 for ovarian cancer, and (iv) clorotoxin to identify glioma.

Similarly CAMLs can be used to determine early recurrence of cancer whenthe cancer was under remission. Currently CT and MRI are used to monitorthe patient's tumor, requiring the tumor to change in size substantiallyto notice the difference. Patients can therefore lose valuable time inbeginning treatment when only subtle size changes occur. CAMLs, alone orin combination with CTCs, can provide early detection of return ofcancer. Non-invasive blood test of CAMLs and CTCs is much lower in costthan CT and MRI.

The capability of tracking CAMLs provides a novel opportunity toroutinely monitor necrosis and chemotherapy or radiation therapyresponse. If the chemotherapy is not working, the CAMLs number will notincrease. This can be used in parallel with CTC detection. If thetreatment is working, the number of pathologically-definable CTCs willdecrease and number of apoptotic CTCs will increase. However, CTCscannot always be detected. If CTCs are detected at the same time asCAMLs, the sensitivity and specificity can be improved. For many cancersthere are large array of chemotherapy agents. If the patient is notresponding to one type of chemotherapy, the patient can quickly switchto another.

The CAMLs can also potentially be used to determine cancer subtyping orgene mutations, translocations or amplification. There are a number ofcancerous nuclei in each CAMLs. Thus, molecular analysis of the nucleusfor genetic mutation, genetic defects, gene translocations can provideinformation to determine treatments. There are drugs that specificallytarget certain gene mutations, translocation or amplifications. CAMLscan be used along or in parallel with CTCs for molecular analysis.

Circulating monocytes have the ability to enter any tissue compartmentof the body, including lymph nodes, bone marrow, most organs, and evencross the blood brain barrier. The detection of CAMLs are not limited toblood, but also can be found in lymph nodes, bone marrow, cerebralspinal fluid, most organs, and urine.

The volume of blood typically used for detection of CTCs is 7.5 mL.Larger volumes of blood will provide more sensitivity and consistency,but smaller volumes such as 3.5 mL may be sufficient. For many CTCdetection methods, larger volumes of blood are not practical for avariety of reasons. However, microfiltration of blood to capture CTCsand/or CAMLs allows more flexibility to increase the sample size. Bloodvolumes of 50 mL have been shown to be successfully screened usingCellSieve™ microfilters with 160,000 pores. The recommended volume ofblood to capture CAMLs would be 7.5 ml or greater.

We claim:
 1. A method of screening a subject for cancer, comprisingdetecting circulating Cancer Associated Macrophage-Like cells (CAMLs) ina biological sample from a subject previously determined to be high riskfor cancer, wherein said detecting comprises: (a) isolating intact cellsof between 20 and 300 micron in size from a biological sample obtainedfrom a subject using a microfilter having pores of about 7-8 micros,wherein the biological sample is peripheral blood, and (b) selectingmulti-nucleated cells isolated in (a) expressing one or more of thefollowing markers: endothelial cell markers CD146, CD202b, and CD31, andmonocyte cell markers CD11c and CD14, wherein antibodies are used toselect cells expressing the one or more markers, thereby detecting CAMLsin a biological sample from a subject, wherein when CAMLs are detectedin the biological sample, the subject is determined to have cancer. 2.The method of claim 1, wherein the cancer is carcinoma or solid tumor.3. The method of claim 1, further comprising detecting circulating tumorcells (CTCs) in the biological sample.
 4. A method for confirming adiagnosing of cancer in a subject, comprising detecting CAMLs in abiological sample from a subject previously diagnosed with cancer,wherein said detecting comprises: (a) isolating intact cells of between20 and 300 micron in size from a biological sample obtained from asubject using a microfilter having pores of about 7-8 microns, whereinthe biological sample is one or more of peripheral blood, blood, andlymph nodes, and (b) selecting multi-nucleated cells isolated in (a)expressing one or more of the following markers: endothelial cellmarkers CD146, CD202b, and CD31, and monocyte cell markers CD11c andCD14, wherein antibodies are used to select cells expressing the one ormore markers, thereby detecting CAMLs in a biological sample from asubject, wherein when CAMLs are detected in the biological sample, thediagnosed of cancer in the subject is confirmed.
 5. The method of claim4, further comprising detecting CTCs in the biological sample, whereinwhen CAMLs and CTCs are detected in the biological sample, the subjectis diagnosed with cancer.
 6. The method of claim 1 or 4, wherein intactcells of between 20 and 300 micron in size are isolated using alow-pressure microfiltration assay.
 7. The method of claim 1 or 4,wherein the cancer is breast, prostate, lung, pancreatic, or colorectal.8. A method for monitoring efficacy of a cancer treatment, comprising(a) determining the number of CAMLs in a biological sample from asubject previously diagnosed with cancer before treatment of the subjectfor cancer, and (b) comparing the number of CAMLs determined in (a) to anumber of CAMLs determined from a similar biological sample from thesame subject at one or more time points after treatment, wherein thenumber of CAMLs in a biological sample is determining by: (a) isolatingintact cells of between 20 and 300 micron in size from a biologicalsample obtained from a subject using a microfilter having pores of about7-8 microns, wherein the biological sample is one or more of peripheralblood, blood, and lymph nodes, and (b) selecting multi-nucleated cellsisolated in (a) expressing one or more of the following markers:endothelial cell markers CD146, CD202b, and CD31, and monocyte cellmarkers CD11c and CD14, wherein antibodies are used to select cellsexpressing the one ore more markers, to obtain CAMLs from the biologicalsample.
 9. The method of claim 8, further comprising (c) determining thenumber of CTCs in the biological sample of (a), and (d) comparing thenumber of CTCs determined in (c) to a number of CTCs determined from thebiological sample of (b).
 10. The method of claim 8, wherein intactcells of between 20 and 300 micron in size are isolated using alow-pressure microfiltration assay.
 11. The method of claim 8, whereinthe cancer is breast, prostate, lung, pancreatic, or colorectal.
 12. Amethod for detecting CAMLs in a biological sample from a subject,comprising: (a) isolating intact cells of between 20 and 300 micron insize from a biological sample obtained from a subject, wherein thebiological sample is peripheral blood, and (b) detecting multi-nucleatedcells isolated in (a) that express one or more of the following markers:endothelial cell markers CD146, CD202b, and CD31, and monocyte cellmarkers CD11c and CD14, wherein antibodies are used to detect cells thatexpress the one or more markers, thereby detecting CAMLs is a biologicalsample from a subject.
 13. The method of claim 12, wherein the subjecthas cancer.
 14. The method of claim 13, wherein the cancer is carcinomaor solid tumor.
 15. The method of claim 12, further comprising detectingCTCs in the biological sample.
 16. The method of claim 12, whereinintact cells of between 20 and 300 micron in size are isolated using oneor more means selected from the group consisting of size exclusionmethodology, red blood cell lysis, FICOLL, a microfluidic chip, and flowcytometry, or a combination thereof.
 17. The method of claim 16, whereinthe size exclusion methodology comprises use of a microfilter.